The development of non-viral vectors for oligonucleotide delivery of plasmid DNA (pDNA), antisense DNA (asDNA), and small interfering RNA (siRNA) remains a substantial scientific challenge. Current methods suffer from low transgene expression levels (pDNA) and problems with transport inside the cell and degradation in the cytoplasm (pDNA, asDNA, siRNA). This research program will test the role of cell plasma membrane disruption in triggering cellular responses that inhibit transfection and/or expression. The structure and dynamics of the nanoscale pores induced in the membrane will be explored and their relationship to the triggering of cellular defense mechanisms determined. The creation of nanoscale pores in the cell membrane will be examined to see if they cause an increase in cytoplasmic nuclease activity. Understanding the cellular responses induced by the non-viral vectors is critical to rational development of these oligonucleotide delivery agents. The specific aims of this research are: 1) Assessment of polymer and polyplex cell membrane disruption 2) Assessment and quantification of the role of plasma membrane permeability in triggering cellular defense mechanisms that inhibit transfection and expression. 3) Design and quantification of polyplex structure. PUBLIC HEALTH RELEVANCE: This work is important to public health because it will uncover the details of cellular mechanism that inhibit efficient use of gene therapies. Gaining understanding of how non-viral vectors activate cell-based defenses against the introduction of foreign oligonucleotides will allow rational optimization of vector design.